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. 2013 May 27;8(5):e65220.
doi: 10.1371/journal.pone.0065220. Print 2013.

Detailed topology mapping reveals substantial exposure of the "cytoplasmic" C-terminal tail (CTT) sequences in HIV-1 Env proteins at the cell surface

Jonathan D Steckbeck  1 , Chengqun Sun, Timothy J Sturgeon, Ronald C Montelaro
Affiliations

Detailed topology mapping reveals substantial exposure of the "cytoplasmic" C-terminal tail (CTT) sequences in HIV-1 Env proteins at the cell surface

Jonathan D Steckbeck et al. PLoS One. .

Abstract

Substantial controversy surrounds the membrane topology of the HIV-1 gp41 C-terminal tail (CTT). While few studies have been designed to directly address the topology of the CTT, results from envelope (Env) protein trafficking studies suggest that the CTT sequence is cytoplasmically localized, as interactions with intracellular binding partners are required for proper Env targeting. However, previous studies from our lab demonstrate the exposure of a short CTT sequence, the Kennedy epitope, at the plasma membrane of intact Env-expressing cells, the exposure of which is not observed on viral particles. To address the topology of the entire CTT sequence, we serially replaced CTT sequences with a VSV-G epitope tag sequence and examined reactivity of cell- and virion-surface Env to an anti-VSV-G monoclonal antibody. Our results demonstrate that the majority of the CTT sequence is accessible to antibody binding on the surface of Env expressing cells, and that the CTT-exposed Env constitutes 20-50% of the cell-surface Env. Cell surface CTT exposure was also apparent in virus-infected cells. Passive transfer of Env through cell culture media to Env negative (non-transfected) cells was not responsible for the apparent cell surface CTT exposure. In contrast to the cell surface results, CTT-exposed Env was not detected on infectious pseudoviral particles containing VSV-G-substituted Env. Finally, a monoclonal antibody directed to the Kennedy epitope neutralized virus in a temperature-dependent manner in a post-attachment neutralization assay. Collectively, these results suggest that the membrane topology of the HIV gp41 CTT is more complex than the widely accepted intracytoplasmic model.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Replacement of HIV gp41 sequences with VSV-G sequences.
The VSV-G epitope tag was used to replace gp41 CTT sequences serially across the length of the CTT as indicated, from CTT1 to CTT14. The VSV-G epitope tag sequence is listed above CTT1.
Figure 2
Figure 2. HIV Env staining of intact live cells.
HEK293T/17 cells transfected with the indicated VSV-G containing Env were stained with (A) anti-HA, and (B) anti-VSV-G monoclonal antibodies. Only intact live cells were selected for analyses of Env exposure to reference monoclonal antibodies. (A) Total cell surface Env expression was determined by staining cells with anti-HA, targeting HA epitope tag inserted into gp120. (B) CTT exposure was determined by staining cells with anti-VSV-G, targeting the VSV-G epitope tag in the CTT sequence as demonstrated in Figure 1. The line at 20% staining is for visual reference only.
Figure 3
Figure 3. CTT reactivity does not transfer from Env-expressing cells to Env-naïve cells.
Transfected cells (mock and CTT3) were stained to determine cell surface anti-VSV-G reactivity (labeled Donor). Clarified supernatants from the Donor cells were transferred to the Recipient cells as described , and the Recipient cells were stained to determine anti-VSV-G reactivity. Only Donor cells expressing CTT3 demonstrated staining with anti-VSV-G MAb. Cell surface CTT was not detected on the Recipient cells incubated with supernatant from CTT3 Donor cells.
Figure 4
Figure 4. Inhibition of HA-specific antibody binding by VSV-G-specific antibody.
Env-expressing cells were first reacted with unlabeled anti-VSV-G (CTT) antibody followed by staining with fluorescently-labeled anti-HA (gp120). The percent reduction in HA-labeled cells compared to HA-stained cells without VSV-G staining is presented as percent HA-inhibition.
Figure 5
Figure 5. CTT exposure can be detected on virus-infected cells.
Unfixed uninfected and HIV-1 89.6-infected CEMx174 cells were surface stained with anti-KE MAb SAR1, and subsequently fixed, permeabilized, and stained with anti-p24 to determine the extent of CTT exposure on virally-infected cells. Dot plots are representative of cells from the live cell population as determined from cellular scatter characteristics.
Figure 6
Figure 6. Relative infectivity of pseudoviruses containing VSV-G-tagged Env.
Supernatants from HEK293T/17 cells cotransfected with pSG3ΔEnv and the indicated Env construct were used to infect TZM-bl cells to determine infectivity of the resulting pseudovirus particles. Results are presented as relative light units (RLU) per 100 µl of transfection supernatant.
Figure 7
Figure 7. Immunoprecipitation of VSV-G tagged pseudoviral particles.
Pseudovirus particles were immunoprecipitated using monoclonal antibodies specific for the VSV-G epitope tag (closed bars) or the gp41 MPER (2F5, open bars). Results are presented as the percent of input p24 that was immunoprecipitated (IP).
Figure 8
Figure 8. Post-attachment neutralization of HIV-1 89.6 by anti-CTT antibody.
Anti-CTT (Kennedy epitope-specific) monoclonal antibody SAR1 was used to determine post-attachment neutralization (PAN) activity at 37°C and 31°C. SAR1 did not exhibit PAN at 37°C, but there was a statistically significant reduction in viral infection when SAR1 was tested for PAN at 31°C. * indicates statistical significance at p<0.05.
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